U.S. patent application number 12/867413 was filed with the patent office on 2011-03-03 for silica magnetic particles having a spherical form and a process for preparing the same.
This patent application is currently assigned to BIONEER CORPORATION. Invention is credited to Jae-Ha Kim, Jong-Hoon Kim, Han-Oh Park, Jong-Gwang Park.
Application Number | 20110054162 12/867413 |
Document ID | / |
Family ID | 41207044 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110054162 |
Kind Code |
A1 |
Kim; Jae-Ha ; et
al. |
March 3, 2011 |
Silica Magnetic Particles Having a Spherical Form and a Process for
Preparing the Same
Abstract
The present invention relates to silica magnetic particles
having a spherical form and a process for preparing the same. The
silica magnetic particles prepared according to the present
invention, which are silica particles that includes the magnetic
particles and additionally have the functional group on the
surfaces, has an advantage that the particle size distribution is
uniform. Further, the silica magnetic particles prepared according
to the present invention can be used as a reagent for separating
biomaterials and a reagent for detecting biomaterials.
Inventors: |
Kim; Jae-Ha; (Daejeon,
KR) ; Park; Jong-Gwang; (Daejeon, KR) ; Kim;
Jong-Hoon; (Daejeon, KR) ; Park; Han-Oh;
(Daejeon, KR) |
Assignee: |
BIONEER CORPORATION
Daejeon
KR
|
Family ID: |
41207044 |
Appl. No.: |
12/867413 |
Filed: |
February 13, 2009 |
PCT Filed: |
February 13, 2009 |
PCT NO: |
PCT/KR2009/000718 |
371 Date: |
November 15, 2010 |
Current U.S.
Class: |
536/25.4 ;
252/62.51R; 427/127 |
Current CPC
Class: |
C01G 49/02 20130101;
B82Y 25/00 20130101; C01B 33/20 20130101; B22F 1/0074 20130101;
C01B 33/12 20130101; C01P 2004/80 20130101; C01P 2004/32 20130101;
C01P 2004/61 20130101; C01P 2004/03 20130101; C01P 2004/84
20130101; H01F 1/0045 20130101; C07H 21/04 20130101; C07H 21/02
20130101 |
Class at
Publication: |
536/25.4 ;
427/127; 252/62.51R |
International
Class: |
C07H 21/00 20060101
C07H021/00; B05D 5/12 20060101 B05D005/12; H01F 1/42 20060101
H01F001/42; C07H 21/02 20060101 C07H021/02; C07H 21/04 20060101
C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 2008 |
KR |
10-2008-0013545 |
Nov 21, 2008 |
KR |
10-2008-0116512 |
Claims
1. A method for preparing silica magnetic particles having a
spherical form comprising: 1) preparing emulsion by adding
surfactant, soluble silicates aqueous solution, and magnetic
particles to a non-polar solvent and then dispersing it using an
ultrasonic wave; and 2) preparing silica magnetic particles by
adding fatty acid to the emulsion.
2. The method according to claim 1, wherein the non-polar solvent
is one or more selected from cyclohexane, hexane, heptane, and
octane.
3. The method according to claim 1, wherein the surfactant is
selected from a group consisting of non-ionic surfactant, cationic
surfactant, and anionic surfactant
4. The method according to claim 3, wherein the non-ionic
surfactant is selected from a group consisting of polyoxyethylene
decyl ether, polyoxyethylene lauryl ether, polyoxyethylene cetyl
ether, polyoxyethylene oleyl ether, polyoxyethylene stearyl ether,
polyoxyethylene octyl decyl ether, polyoxyethylene tridecyl ether,
polyoxyethylene nonylphenol ether, polyoxyethylene octylphenol
ether, polyoxyethylene phenyl ether, polyoxyethylene sorbitan
ester, sorbitan monolaurate, sorbitan monopalmitate, sorbitan
monosterate, sorbitan trioleate, polyoxyethylene glycol,
polyoxyethylene oleyl ester, and mixtures thereof; the cationic
surfactant is selected from a group consisting of dodecyl ammonium
chloride, cetyltrimethylammonium bromide, alkylammonium
methosulfate, alkyl dimethyl ammonium chloride, and mixtures
thereof; the anionic surfactant is selected from a group consisting
of sodium stearate, sodium laurate, sodium palmitate, potassium
stearate, potassium laurate, potassium palmitate, sodium lauryl
sulfate, sodium dodecylbenzene sulfonate, and mixtures thereof.
5. The method according to claim 3, wherein the surfactant is used
as content of 5 to 30 parts by weight for 100 parts by weight of a
non-polar solvent.
6. The method according to claim 5, wherein the surfactant is the
non-ionic surfactant and is used as content of 10 to 25 parts by
weight for 100 parts by weight of a non-polar solvent.
7. The method according to claim 6, wherein the surfactant is
polyoxyethylene nonylphenol ether.
8. The method according to claim 1, wherein soluble silicates
aqueous solution is selected from sodium silicates aqueous
solution, potassium silicates aqueous solution, or lithium
silicates aqueous solution.
9. The method according to claim 8, wherein the soluble silicates
aqueous solution is used as content of 1 to 20 parts by weight for
100 parts by weight of a non-polar solvent.
10. The method according to claim 9, wherein the soluble silicates
aqueous solution is the sodium silicates aqueous solution and is
used as content of 1 to 10 parts by weight for 100 parts by weight
of a non-polar solvent.
11. The method according to claim 8, wherein the concentration of
the soluble silicates aqueous solution is 0.1 mol/L to 5 mol/L.
12. The method according to claim 1, wherein the magnetic particles
is one or more particles selected from a group consisting of iron
oxide, ferrite, iron, cobalt, manganese, chromium, nickel, zinc,
and mixtures thereof.
13. The method according to claim 12, wherein the magnetic
particles are used as content of 0.01 to 1.0 parts by weight for
100 parts by weight of a non-polar solvent.
14. The method according to claim 13, wherein the magnetic
particles are iron oxide particles and are used as content of 0.01
to 0.5 parts by weight for 100 parts by weight of a non-polar
solvent.
15. The method according to claim 1, wherein fatty acid is one or
more selected from compounds according to the following Formula 1.
R--COOH [Formula 1] (In the above Formula 1, R is selected from
straight or branched alkyl group of C8.about.C20 and the alkyl
group includes a double bond of 1 or more or a triple bond of 1 or
more within a carbon chain and is further substituted with a
hydroxy group).
16. The method according to claim 15, wherein the fatty acid is
selected from a group consisting of myristic acid, palmitic acid,
lauric acid, stearic acid, linoleic acid, linolenic acid,
ricinoleic acid, oleic acid, and mixtures thereof.
17. The method according to claim 16, wherein the fatty acid is
used as content of 0.1 to 10 parts by weight for 100 parts by
weight of a non-polar solvent.
18. The method according to claim 16, wherein the fatty acid is
myristic acid and used as content of 1 to 5 parts by weight for 100
parts by weight of a non-polar solvent.
19. The method according to claim 1, further comprising a step of
introducing functional groups on the surfaces of the silica
magnetic particles after the step 2.
20. The method according to claim 19, wherein the functional group
is one or more selected from an amine group, a carboxylic acid
group, an epoxy group, a (C1.about.C30) alkyl group, a streptavidin
group, a biotin group, and an iminodiacetic acid group.
21. The method according to claim 20, wherein the amine group is
introduced from aminopropyldiisopropylethoxysilane,
aminopropyltrimethoxysilane, aminopropyltriethoxysilane,
aminopropylmethyldiethoxysilane, aminophenyltrimethoxysilane,
phenylaminopropyltrimethoxysilane,
aminoethylaminopropyltrimethoxysilane,
aminoethylaminopropyltriethoxysilane,
aminoethylaminopropylmethyldimethoxysilane,
aminoethylaminoisobutylmethyldimethoxysilane,
trimethoxysilylpropyldiethylenetriamine, or mixtures thereof; the
carboxylic acid group is introduced from
trimethoxysilylpropylethylenediamine triacetic acid or introduced
by introducing the amine group into the silica magnetic particles
and then reacting it with succinic anhydride; the epoxy group is
introduced from glycidoxypropyltrimethoxysilane,
glycidoxypropyltriethoxysilane,
glycidoxypropylmethyldimethoxysilane,
glycidoxypropylmethyldiethoxysilane,
glycidoxypropyldimethylmethoxysilane,
glycidoxypropyldimethylethoxysilane,
epoxycyclohexylethyltrimethoxysilane, or mixtures thereof; the
(C1.about.C30) alkyl group is introduced from trimethoxy
(C1.about.C30) alkylsilane, triethoxy (C1.about.C30) alkylsilane
(triethoxyoctadecylsilane), or mixtures thereof; the streptavidin
group is introduced by introducing the amine group into the silica
magnetic particles and then reacting it with streptavidin; the
biotin group is introduced by introducing the amine group into the
silica magnetic particles and then reacting it with biotin; the
iminodiacetic acid group is introduced by introducing the epoxy
group into the silica magnetic particles and then reacting it with
iminodiacetonitrile.
22. Silica magnetic particles having a spherical form prepared from
the preparing method of claim 1.
23. A method for separating biomaterials, comprising: forming
complexes of silica magnetic particles having a spherical form and
biomaterials by inducing the bonding of the silica magnetic
particles having a spherical form according to claim 22 and the
biomaterials; separating the complexes using external magnetic
field; and obtaining the biomaterials from the separated
complexes.
24. The method for separating biomaterials according to claim 23,
wherein the biomaterial is nucleic acid.
25. The method for separating biomaterials according to claim 24,
wherein the nucleic acid is selected from a group consisting of
plasmid DNA, genomic DNA, cDNA, PCR DNA (polymerase chain reaction
DNA), RNA, siRNA, ribozymes, aptamers, and oligonucleotide.
Description
TECHNICAL FIELD
[0001] The present invention relates to silica magnetic particles,
a process for preparing the same, and a method for separating
biomaterials using the silica magnetic particles.
BACKGROUND ART
[0002] In recent years, a study on a material for bio related
research and bio medical service using magnetic substances has been
frequently attempted. In particular, studies on a material for
separating biomaterials and a use as a medical material have been
actively attempted. Among others, a study on magnetic functional
silane coated particles where organic functional compounds are
coated on magnetic particles has been progressed so that the
magnetic functional silane coated particles can be used for
separation and purification of DNA and RNA, separation and
purification of protein and amino acid, a bio sensor, a drug
delivery system, magnetic resonance imaging (MRI) contrast agent,
local warm-heating treatment, etc.
[0003] The magnetic particles start to be frequently applied as
basic materials used in biotechnology research and have been used
to quickly and simply separate the biomaterials. A method for
separating biomaterials, in particular, nucleic acid or protein
according to the related art needs much time and labor force since
it should perform several extracting and centrifugal separating
steps but degrades the yield and purity of the separated
biomaterials and is not suitable for use as a method for automation
or mass separation. However, in recent research, special magnetic
particles were prepared and a method for very quickly and
efficiently separating biomaterials using magnetic particles under
appropriate buffer conditions was developed (U.S. Pat. No.
5,523,231, and U.S. Pat. No. 5,665,554).
[0004] Further, when using the above-mentioned method for
separating biomaterials, an automation method, which can
simultaneously process many samples and separate the biomaterials,
can be provided. For example, when using a robot automatic
apparatus, several hundreds or several thousands of samples can be
automatically processed and desired biomaterials can be separated
from the samples in large quantities.
[0005] In order to discriminate nucleic acid or protein from
several cell mixtures, an effective and reproducible separating
method is needed, and then a separating method using magnetic
particles has been recently developed.
[0006] The separation of nucleic acid (DNA and RNA) from the
biological samples is the most important step in a biochemistry
research and diagnostic process.
[0007] If genetic materials (nucleic acid) are not separated from
the samples, genetic detection, genetic cloning, genetic
sequencing, genetic amplification, cDNA synthesis, and the like,
which are subsequent steps, can not be performed. A method for
separating nucleic acid using magnetic particles is a separating
method that induces a bonding with biomaterials using the magnetic
particles and then applies external magnetic field to samples,
wherein it is known that the proper size of the magnetic particles
used for separating and purifying DNA, RNA, protein, etc., is
generally approximately 500 to 2000 nm.
[0008] As such, in order for the magnetic particles to be used for
the separation and purification of gene (nucleic acid) or protein,
it is preferable that they should have magnetic properties as well
as should be conjugated with a functional group that bonds a gene
or a specific protein on the surfaces of the particles. To this
end, there is a need to coat the magnetic particles using the
organic functional group or silica.
[0009] Among the magnetic particles used for separating the
above-mentioned biomaterials, iron oxide particles are
representative. The magnetic iron oxide particles generally exist
as magnetite (Fe.sub.3O.sub.4), maghemite (Fe.sub.2O.sub.3), or
hematite (Fe.sub.2O.sub.3) and the magnetic iron oxide particles
can be used to separate and purify biomaterials, for example,
nucleic acid (DNA and RNA), separate and purify protein, purify
peptide and polypeptide, purify lipids, and the like.
[0010] The methods for preparing magnetic particles for separating
biomaterials according to the related art can prepare the magnetic
particles without agglomeration and interaction between the
magnetic particles only by preparing magnetic iron or iron oxide
particles from iron salt compounds using a liquid-phase reduction
method and coating the prepared magnetic iron or iron oxide
particles with silica, polymer or gold or silver, which are a
non-magnetic materials. In recent years, among these magnetic
particles, silica magnetic particles were being mainly developed
(U.S. Pat. No. 6,027,945, U.S. Pat. No. 6,673,631, U.S. Pat. No.
7,183,002, and JP Patent No. 3253638). However, the silica magnetic
particles has disadvantages in that a preparing process is
complicated, the particle form is uneven, the separation yield in
the separation and purification of biomaterials such as nucleic
acid, and the like is degraded.
[0011] Meanwhile, a method for preparing silica magnetic particles
having a spherical form from a method that forms a W/O type
emulsion using sodium silicates aqueous solution and emulsifier,
adds ammonium sulfate aqueous solution thereto, and then
sufficiently agitates it is known in JP Laid-Open Patent No.
2001-136970 and 1994-047273. However, since the above method
requires a process of dispersing the ammonium sulfate aqueous
solution in the W/O type emulsion and a process of performing a
sufficient agitation using an ultrasonic wave, etc., so that the
dispersed ammonium sulfate aqueous solution micelle reacts with the
sodium silicates aqueous solution micelle, the preparing process is
complicated as well as since the size of the sodium silicates
aqueous solution micelle is changed in the agitating process with
the ammonium sulfate aqueous solution, it is difficult to uniformly
control the silica particle size distribution to be formed.
[0012] KR Laid-Open Patent No. 2006-0061494 about the magnetic
functional silica coated particles discloses a method for preparing
magnetic functional silica coated particles by introducing an amine
group or a chloro group on a surface as a method for preparing
magnetic functional silica coated particles for separating and
purifying nucleic acid, DNA, and RNA. However, the preparing method
of the above-mentioned patent has disadvantages in that very
expensive tetraethyl orthosilicate (TEOS) is used, the preparing
process is complicated, and the particles are non-uniform. Further,
KR Registration Patent No. 0541282 discloses a method that modifies
the magnetic nanoparticles with silane materials and uses it;
however, since the used magnetic nanoparticles themselves are
magnetic substances, it has problems, such as biotoxicity, etc.
DISCLOSURE OF INVENTION
Technical Problem
[0013] The present invention proposes to solve the problems in the
related art. An object of the present invention is to provide a
novel preparing method that can simplify a preparing process and
uniformly control the size of silica magnetic particles, as
compared to a method for preparing silica magnetic particles in the
related art.
[0014] More specifically, an object of the present invention is to
a preparing method that can simplify a preparing process, easily
induce various functional groups, make prepared silica magnetic
particles in a spherical form, and uniformly control the size of
silica magnetic particles, by using an emulsion method and adding
fatty acid that can be dissolved in non-polar solvents to induce
sol-gel reaction in a method for chemically preparing the silica
magnetic particles used for separating biomaterials.
[0015] Further, another object of the present invention is to
provide a method for preparing magnetic functional silica coated
particles that can be effectively used for a reagent for separating
or purifying biomaterials or for detecting biomaterials by
introducing various functional groups on silica magnetic particles
having a spherical form prepared from the preparing method.
Technical Solution
[0016] The present invention relates to a method for preparing
silica magnetic particles having a spherical form that uses an
emulsion method and adds fatty acid that can be dissolved in
non-polar solvents to induce sol-gel reaction, silica magnetic
particles having a spherical form with a uniform particle size
distribution prepared from the same, and a method for separating
and purifying biomaterials using the silica magnetic particles
having the spherical form.
[0017] The present invention provides a method for preparing silica
magnetic particles having a spherical form including the following
steps.
[0018] 1) preparing emulsion by adding surfactant, soluble
silicates aqueous solution, and magnetic particles to a non-polar
solvent and then dispersing it using an ultrasonic wave; and
[0019] 2) preparing silica magnetic particles by adding fatty acid
to the emulsion.
[0020] Further, the present invention provides silica magnetic
particles having a spherical form, which is prepared according to
the above preparing method, containing the magnetic particles
therein. The silica magnetic particles having a spherical form
prepared according to the preparing method of the present invention
are shown in FIG. 1 and FIGS. 4 to 6, it can be confirmed from FIG.
1 and FIGS. 4 to 6 that the size of the prepared silica magnetic
particles having a spherical form is 1 to 20 .mu.m, and referring
to the particle size analyzing results of FIG. 2, it can be
appreciated that the particle size distribution is uniform.
[0021] A method for preparing silica magnetic particles further
includes additionally the step of introducing a functional group on
surfaces of the silica magnetic particles prepared in the step 2),
after the step 2). The introduction of the functional group can be
made through the bonding reaction with compounds for introducing
the functional group on the silica magnetic particles by dispersing
the silica magnetic particles having a spherical form prepared in
the step 2) in the solvents and contacting it with compounds for
introducing the functional group.
[0022] Preferably, the functional group is one or more selected
from an amine group, a carboxylic acid group (--COOH), an epoxy
group, a (C1.about.C30) alkyl group, a streptavidin group, a biotin
group, and an iminodiacetic acid group.
[0023] The silica magnetic particles prepared in the step 2) has a
hydroxyl group (--OH) group on the surface thereof, such that they
can be used for separating the biomaterials, specifically, nucleic
acid. According to the concrete embodiment, a method for separating
the nucleic acid can be provided by using the silica magnetic
particles having a spherical form prepared in the step 2) as a
nucleic acid bonding carrier and using a reagent for bonding the
silica magnetic particles having a spherical form with the nucleic
acid, for example, a chaotropic reagent, etc.
[0024] The silica magnetic particles having a spherical form
prepared through the step of introducing the functional group can
be used for a reagent for widely separating or purifying the
biomaterials or for detecting the biomaterials through various
functional groups.
[0025] Hereinafter, the contents of the present invention will be
described in detail.
[0026] In the present invention, a method for preparing silica
magnetic particles having a spherical form for separating
biomaterials includes a first step of preparing emulsion by adding
surfactant, soluble silicates aqueous solution, and magnetic
particles to a non-polar solvent and then dispersing it using an
ultrasonic wave, a second step of preparing silica magnetic
particles by adding fatty acid to the emulsion and by forming
silica by sol-gel reaction, and a third step of preparing
functional silica magnetic particles by adding functional compounds
to the silica magnetic particles.
[0027] In the present invention, the first step for preparing the
silica magnetic particles having a spherical form prepares emulsion
by adding surfactant, soluble silicates aqueous solution, and
magnetic particles to a non-polar solvent and then dispersing it
using an ultrasonic wave. When adding an appropriate amount of
soluble silicates aqueous solution and surfactant to the non-polar
solvent and processing it using the ultrasonic wave, the micelle is
formed by the surfactant. Herein, the prepared micelle, which is
inverse emulsion, is a form where an aqueous solution micelle is
formed on oil that is the non-polar solvent, that is, W/O type
emulsion.
[0028] Further, the added magnetic particles exist in the micelle
by the ultrasonic wave. The micelle having a spherical form
including the soluble silicates aqueous solution reacts with the
fatty acid added later to completely prepare the silica having a
spherical form. The first step of the preparing method according to
the present invention is a step that forms the soluble silicates
aqueous solution having a uniform size and disperses the magnetic
particles so that the magnetic particles exist in the micelle.
[0029] In the preparing method according to the present invention,
the non-polar solvent is a solvent that can prepare the W/O type
emulsion as a solvent having low solubility to water, specifically,
can use a solvent having solubility of 8% or less to water. It is
preferable that as the non-polar solvent, cyclohexane, hexane,
heptane, octane or mixtures thereof are used; however, it is not
limited thereto.
[0030] It is preferable that as the materials, which can be used as
the surfactant in the preparing method according to the present
invention, non-ionic surfactant, cationic surfactant, or anionic
surfactant is used, but more preferably, the non-ionic surfactant
is used. The surfactant can be used as content of 5 to 30 parts by
weight for 100 parts by weight of a non-polar solvent, preferably
as content of 10 to 25 parts by weight. When the surfactant exceeds
30 parts by weight, it affects the formation of emulsion so that
the silica having a spherical form is not prepared. When the
surfactant is less than 5 parts by weight, the number of micelles
is too small so that the yield of silica magnetic particles having
a spherical form is too low, thereby causing the problem in
productivity.
[0031] In detail, it is preferable that the non-ionic surfactant is
polyoxyethylene decyl ether, polyoxyethylene lauryl ether,
polyoxyethylene cetyl ether, polyoxyethylene oleyl ether,
polyoxyethylene stearyl ether, polyoxyethylene octyl decyl ether,
polyoxyethylene tridecyl ether, polyoxyethylene nonylphenol ether,
polyoxyethylene octylphenol ether, polyoxyethylene phenyl ether,
polyoxyethylene sorbitan ester, sorbitan monolaurate, sorbitan
monopalmitate, sorbitan monosterate, sorbitan trioleate,
polyoxyethylene glycol, polyoxyethylene oleyl ester, or mixtures
thereof; however, it is not limited thereto. It is preferable that
the cationic surfactant is dodecyl ammonium chloride,
cetyltrimethylammonium bromide, alkylammonium methosulfate, alkyl
dimethyl ammonium chloride, or mixtures thereof; however it is not
limited thereto. It is preferable that the anionic surfactant is
sodium stearate, sodium laurate, sodium palmitate, potassium
stearate, potassium laurate, potassium palmitate, sodium lauryl
sulfate, sodium dodecylbenzene sulfonate, or mixtures thereof;
however, it is not limited thereto.
[0032] In the preparing method according to the present invention,
as the soluble silicates aqueous liquid, preferably, sodium
silicates aqueous solution, potassium silicates aqueous solution,
or lithium silicates aqueous solution is used, but more preferably,
the sodium silicates aqueous solution is used; however, it is not
limited thereto. It is preferable that the morality M of the
soluble silicates aqueous liquid is 0.1 mol/L to 5 mol/L.
[0033] When the morality is less than 0.1 mol/L, the reaction speed
is too slow, thereby causing the problem in the processes and when
the morality is too high exceeding 5 mol/L, the reaction speed is
too rapid, thereby causing a problem in that silica having a
uniform spherical form is not formed.
[0034] Preferably, the soluble silicates aqueous solution is used
as content of 1 to 20 parts by weight for 100 parts by weight of a
non-polar solvent, more preferably as content of 1 to 10 parts by
weight. When the soluble silicates aqueous solution exceeds 20
parts by weight, the micelle is too large in the emulsion so that
the silica magnetic particles are too largely prepared. When the
soluble silicates aqueous solution is less than 1 parts by weight,
the micelle of the surfactant is not formed in the emulsion,
thereby causing a problem in that the silica magnetic particles
having a spherical form is not prepared.
[0035] In the preparing method according to the present invention,
it is preferable that the material, which can be used as the
magnetic particles is one or more selected from iron oxide
(hematite, maghemite; Fe.sub.2O.sub.3, magnetite; Fe.sub.3O.sub.4),
ferrite, iron, cobalt, manganese, chromium, nickel, zinc, or
mixtures thereof; however, it is not limited thereto. It is most
preferable that iron oxide (magnetite) having a size of 100 to 300
nm is used. The magnetic iron oxide particles, which are directly
prepared or sold, can be used. The method for preparing magnetic
iron oxide particles can prepare iron oxide by preparing magnetic
iron particles while generating carbon monoxide by pyrolysis at the
time of instantly injecting carbonyl iron in a high-temperature
solvent and then oxidizing the prepared magnetic iron particles. As
another preparing method, a method for preparing iron oxide by
adding ammonia water to a mixing solution of FeCl.sub.2 and
FeCl.sub.3 is widely known.
[0036] The magnetic particles can be used as content of 0.01 to 1.0
parts by weight for 100 parts by weight of a non-polar solvent,
preferably as content of 0.01 to 0.5 parts by weight. When the
magnetic particles exceed 1.0 parts by weight, it exists out of the
micelle of the emulsion solution, thereby causing a problem in that
silica having a spherical form can not be prepared.
[0037] When the magnetic particles are less than 0.01 parts by
weight, the number of magnetic substances included in the silica
particles is too small, thereby causing a problem in that magnetism
for magnetic force is reduced.
[0038] In the preparing method according to the present invention,
the second step prepares the silica magnetic particles having a
spherical form by adding fatty acid that can be dissolved in the
non-polar solvent while agitating the emulsion solution prepared in
the first step and performing the reaction for forming the silica
of the micelle. The fatty acid is directly added to the emulsion or
the fatty acid solution, which is dissolved in the non-polar
solvent, can be added thereto. It is preferable that a time to add
the fatty acid or the fatty acid solution is 10 to 30 minutes and
it is advantageous that the agitation is performed at speed as
quickly as possible so as to improve the reaction speed for forming
the silica. However, when the agitation speed is too quick, since
the uniformity of the size of the produced silica magnetic
particles can be degraded, it is preferable that the agitation
speed is in the range of about 50 to 2000 rpm.
[0039] If the fatty acid can be dissolved in the non-polar solution
and has acidity, any fatty acid can be used. Further, all of a
natural fatty acid and a synthesized fatty acid can be used.
Specifically, the fatty acid may include compounds according to the
following Formula 1 or mixtures thereof, for example.
R--COOH [Formula 1]
[0040] (In the above Formula 1, R is selected from straight or
branched alkyl group of C8.about.C20 and the alkyl group can
include a double bond of 1 or more or a triple bond of 1 or more
within a carbon chain and can be further substituted with a hydroxy
group).
[0041] An example of the fatty acid usable in the preparing method
according to the present invention may include myristic acid,
palmitic acid, lauric acid, stearic acid, linoleic acid, linolenic
acid, ricinoleic acid, oleic acid, or mixtures thereof. The fatty
acid can be used as content of 0.1 to 10 parts by weight for 100
parts by weight of a non-polar solvent, preferably as content of 1
to 5 parts by weight. When the fatty acid exceeds 10 parts by
weight, it affects the formation of emulsion micelle, thereby
causing a problem in that the silica having a spherical form is not
prepared. When the fatty acid is less than 0.1 parts by weight,
there is a problem in that the reaction for synthesizing the silica
particles is not completely progressed in the emulsion
solution.
[0042] Further, the preparing method according to the present
invention may further include filtering, washing, and drying after
the second step. The filtering uses a microfilter paper. The
washing is repetitively performed several times using ethanol and
ultra pure water. The drying is performed on the prepared silica
magnetic particles having a spherical form at a temperature of 100
to 300.degree. C., preferably 120.degree. C. to 200.degree. C. for
2 hours or more in a drying apparatus.
[0043] As an example according to the present invention,
photographs of a scanning electron microscope for the prepared
silica magnetic particles having a spherical form are shown FIG. 1
and FIGS. 4 to 6 and the particle size analyzing results are shown
in FIG. 2. Further, referring to FIG. 1, FIG. 2, and FIGS. 4 to 6,
in the preparing method according to the present invention, it can
be confirmed that the form of the silica magnetic particles
prepared by subjecting to the step 2) is a spherical form and the
size of the particles is uniformly prepared in the range of 1 to 20
.mu.m. Further, referring to zeta potential analyzing results of
FIG. 3, it can be confirmed that there is a hydroxy group (--OH) on
the surfaces of the silica magnetic particles having a spherical
form. In other words, when the zeta potential is measured, it
indicates a negative value due to the hydroxy group on the surface,
wherein the value is in the range of -30 mV to -60 mV as shown in
FIG. 3.
[0044] On the other hand, as a comparative example, when the silica
magnetic particles are prepared using ammonium sulfate aqueous
solution instead of the fatty acid, since the size of particles are
non-uniform as shown in FIG. 7, it is confirmed that the
reproducibility is degraded due to the repetition of the preparing
process such that it is difficult to uniformly control the size of
particles.
[0045] In the preparing method according to the present invention,
the step 3) is a step that prepares the functional silica magnetic
particles having a spherical form where the silica magnetic
particles having a spherical form prepared in the step 2) contact
compounds for introducing various functional groups so as to
introduce the functional groups. The step 3) can be performed by
dispersing the silica magnetic particles having a spherical form
prepared in the step 2) in a solvent and then contacting them with
the compounds for introducing the functional groups and performing
the reaction that bonds the compounds for introducing the
functional groups on the surfaces of the silica magnetic particles.
Since the hydroxy group (--OH) is formed on the surfaces of the
silica magnetic particles having a spherical form prepared in the
step 2), the bonding reaction with the compounds for introducing
various functional groups can be made. The solvent is not limited
in the reaction of the step 3) and water, hydrocarbon solvent,
halogenated hydrocarbon solvent, etc. can be used alone or in
combinations thereof.
[0046] In the present invention, as a kind of the functional group,
there is one or more selected from an amine group, a carboxylic
acid group, an epoxy group, a (C1.about.C30) alkyl group, a
streptavidin group, a biotin group, and an iminodiacetic acid;
however, it is not necessarily limited thereto.
[0047] In the preparing method according to the present invention,
compounds for introducing an amine group can use silane compounds
having an alkyl group where at least one of amine groups
(--NH.sub.2,--NH--,
##STR00001##
is substituted. As the silane compounds,
aminopropyldiisopropylethoxysilane, aminopropyltrimethoxysilane,
aminopropyltriethoxysilane, aminopropylmethyldiethoxysilane,
aminophenyltrimethoxysilane, phenylaminopropyltrimethoxysilane,
aminoethylaminopropyltrimethoxysilane,
aminoethylaminopropyltriethoxysilane,
aminoethylaminopropylmethyldimethoxysilane,
aminoethylaminoisobutylmethyldimethoxysilane,
trimethoxysilylpropyldiethylenetriamine, or mixtures thereof can be
used; however, it is not necessarily limited thereto.
[0048] In the preparing method according to the present invention,
it is preferable that the compounds for introducing carboxylic acid
is trimethoxysilylpropylethylenediamine triacetic acid; however, it
is not necessarily limited thereto. The carboxylic acid can be
introduced by reacting dicarboxylic acid anhydride such as succinic
anhydride, etc., with the silica magnetic particles into which the
amine group is introduced.
[0049] In the preparing method according to the present invention,
as the compounds for introducing an epoxy group, there are silane
compounds having a glycidoxy group or an epoxy group. Specifically,
glycidoxypropyltrimethoxysilane, glycidoxypropyltriethoxysilane,
glycidoxypropylmethyldimethoxysilane,
glycidoxypropylmethyldiethoxysilane,
glycidoxypropyldimethylmethoxysilane,
glycidoxypropyldimethylethoxysilane,
epoxycyclohexylethyltrimethoxysilane, or mixtures thereof can be
used; however, it is not necessarily limited thereto.
[0050] In the preparing method according to the present invention,
the compounds for introducing a (C1.about.C30) alkyl group can use
silane compounds having the (C1.about.C30) alkyl group.
Specifically, trimethoxy (C1.about.C30) alkylsilane, triethoxy
(C1.about.C30) alkylsilane, or mixtures thereof can be used. It is
preferable that as the compounds corresponding thereto,
trimethoxyoctadecylsilane, triethoxyoctadecylsilane, or mixtures
thereof are used; however, it is not necessarily limited
thereto.
[0051] In the preparing method according to the present invention,
the streptavidin group can be introduced by reacting streptavidin
with the silica magnetic particles into which the amine group is
introduced; however, it is not limited thereto.
[0052] In the preparing method according to the present invention,
the biotin group can be introduced by reacting biotin with the
silica magnetic particles into which the amine group is introduced;
however, it is not limited thereto.
[0053] In the preparing method according to the present invention,
the iminodiacetic acid group can be introduced by reacting
iminodiacetonitrile with the silica magnetic particles into which
the epoxy group is introduced; however, it is not limited
thereto.
[0054] In the functional silica magnetic particles into which the
functional group prepared according to the preparing method of the
present invention is introduced, magnetic particles having a size
of several tens to several hundreds nanometer are enclosed by
silica and the functional group layer inducing various functional
groups are formed on the silica surfaces. In the case where the
functional group is amine groups (--NH.sub.2, --NH--,
##STR00002##
when the zeta potential is measured, it indicates a positive value.
When the amine group according to the seventh embodiment is
introduced, the value indicates values of +20 mV to +50 mV as shown
in FIG. 8. In the case of the functional silica magnetic particles
into which carboxylic acid is introduced, when the zeta potential
is measured, it indicates a negative value due to a carboxylic acid
group (COOH), that is, values of -30 mV to -60 mV. In the case of
the functional silica magnetic particles into which the epoxy group
or the (C1.about.C30) alkyl group is introduced, since the
functional group does not have potential, the potential does not
appear. In the case of the functional silica magnetic particles
into which a streptavidin group is introduced, when the zeta
potential is measured, it indicates a negative value due to the
streptavidin, that is, indicates values of -30 mV to -60 mV.
[0055] In the case of the functional silica magnetic particles into
which the biotin group is introduced, since the biotin does not
have potential, the potential does not appear. In the case of the
functional silica magnetic particles into which the iminodiacetic
acid is introduced, when the zeta potential is measured, it
indicates a negative value due to two carboxylic acid groups
(COOH), that is, values of -30 mV to -60 mV.
[0056] The silica magnetic particles having a spherical form
prepared according to the preparing method according to the present
invention can be used for separating the biomaterials in various
forms.
[0057] Specifically, the method for separating the biomaterials
according to the present invention includes the following preparing
steps:
[0058] forming complexes of silica magnetic particles having a
spherical form and biomaterials by inducing the bonding of the
silica magnetic particles having a spherical form and the
biomaterials;
[0059] separating the complexes using external magnetic field;
and
[0060] obtaining the biomaterials from the separated complexes.
[0061] As the biomaterials, there are plasmid DNA, genomic DNA,
cDNA, PCR DNA (polymerase chain reaction DNA), RNA, siRNA,
ribozymes, aptamers, oligonucleotide, DNA primers, protein,
peptide, polypeptide, amino acid, recombinant protein, antibody,
lipids, or cells, and the like; however, it is not necessarily
limited thereto.
[0062] Generally, in order for the magnetic particles to be used
for separating the nucleic acid, the magnetic particles are coated
with silica.
[0063] The method for separating the nucleic acid using the silica
separates the nucleic acid using chaotropic reagent, which is a
widely known method (R. Boom et al., J. Clin. Microbiol., Vol
28(3), p 495-503 (1990)). If the magnetic particles are coated with
silica, they are bonded with the nucleic acid using the chaotropic
reagent and the silica magnetic particles are separated using
external magnetic force, thereby separating the nucleic acid.
[0064] The silica magnetic particles having a spherical form
prepared by the preparing method according to the present invention
can be used for separating the nucleic acid in various forms. As
the nucleic acid, there are plasmid DNA, genomic DNA, cDNA, PCR DNA
(polymerase chain reaction DNA), RNA, siRNA, ribozymes, aptamers,
oligonucleotide, DNA primers, and the like.
[0065] The method for separating and purifying the nucleic acid
using the silica magnetic particles having a spherical form
according to the present invention is as follows. The first step
bonds the nucleic acid to the silica magnetic particles by mixing
the silica magnetic particles having a spherical form according to
the present invention with samples including the nucleic acid to be
separated. At this time, a binding buffer can be used. An example
of the binding buffer may include the chaotropic reagent. As the
chaotropic reagent, there are guanidine salt, urea, chloride,
iodide, perchlorate, (iso)thiocyanate, and the like. As the
concrete compound, there are sodium perchlorate, guanidine
hydrochloride, guanidine isothiocyanate, potassium iodide,
potassium thiocyanate, sodium chloride, sodium isothiocyanate,
magnesium chloride, sodium iodide, and the like; however, it is not
limited thereto. It is preferable that the chaotropic reagent is
used at a concentration of 1 to 8 M (mol/L).
[0066] The second step of separating the nucleic acid, which is a
step that separates the silica magnetic particles to which the
nucleic acid is bonded, collects the silica magnetic particles to
which the nucleic acid is bonded in a wall surface of a container
by external magnetic force and separates and washes the remaining
materials that are not bonded.
[0067] The third step, which is a step that removes the external
magnetic force and separates the nucleic acid from the silica
magnetic particles to which the nucleic acid is bonded, separates
the nucleic acid to which the silica magnetic particles are bonded
by using an elusion buffer (tris-(hydroxymethyl)amino methane
buffer).
ADVANTAGEOUS EFFECTS
[0068] The silica magnetic particles having a spherical form
prepared according to the present invention includes the magnetic
particles, has an advantage in that the particle size distribution
of silica having the functional group on the surface is uniform and
the particle is a spherical form, and can be used as the reagent
for separating the biomaterials and for detecting the
biomaterials.
BRIEF DESCRIPTION OF DRAWINGS
[0069] The above and other objects, features and advantages of the
present invention will become apparent from the following
description of preferred embodiments given in conjunction with the
accompanying drawings, in which:
[0070] FIG. 1 is photographs of a scanning electron microscope
(SEM) for silica magnetic particles having a spherical form
prepared according to Example 1;
[0071] FIG. 2 is particle size analyzing results of the silica
magnetic particles having a spherical form prepared according to
Example 1;
[0072] FIG. 3 is zeta potential analyzing results of the silica
magnetic particles having a spherical form prepared according to
Example 1;
[0073] FIG. 4 is photographs of a scanning electron microscope
(SEM) for silica magnetic particles having a spherical form
prepared according to Example 4;
[0074] FIG. 5 is photographs of a scanning electron microscope
(SEM) for silica magnetic particles having a spherical form
prepared according to Example 5;
[0075] FIG. 6 is photographs of a scanning electron microscope
(SEM) for silica magnetic particles having a spherical form
prepared according to Example 6;
[0076] FIG. 7 FIG. 4 is photographs of a scanning electron
microscope (SEM) for silica magnetic particles having a spherical
form prepared according to Comparative Example 1; and
[0077] FIG. 8 is zeta potential analyzing results of silica
magnetic particles having a spherical form into which an amine
group prepared according to Example 7 is introduced.
BEST MODE FOR CARRYING OUT THE INVENTION
[0078] Hereinafter, the present invention will be described in
detail with reference to examples. However, the following examples
illustrate the present invention, by way of example only and the
contents of the present invention are not limited to the following
examples.
Example 1
Preparing of Silica Magnetic Particles Having a Spherical Form
Using Myristic Acid
[0079] 400 ml of cyclohexane is put in a 2 L flask and 48 ml of
polyoxyethylene nonylphenol ether (Dongnam Chem., Korea, MONOPOL NP
1018, number average molecule weight (M.sub.n): 585) that is
surfactant and 16 mL of sodium silicate aqueous solution (1.5M
concentration) are put therein. Thereafter, it is dispersed for 30
minutes using an ultrasonic wave. 0.4 g of iron oxide magnetic
particles (magnetite) (SEAHAN, Korea, SMT-02H, average particle
size: 300 nm) is put in the solution and dispersed for 30 minutes
using an ultrasonic wave. 5.5 g of myristic acid is dissolved in 40
ml of cyclohexane to prepare myristic acid solution and then, the
myristic acid solution is dropped for 30 minutes while agitating
emulsion solution. After the dropping, the reaction is completely
performed by agitating it at 300 rpm under room temperature for 2
hours to obtain silica magnetic particles.
[0080] After the reaction completes, products in a reactor are
separated by a filter and washed twice using ethanol and ultra pure
water. The obtained silica magnetic particles are put in a drier
and dried at 160.degree. C. for 2 hours.
[0081] Analysis results of a scanning electron microscope (SEM) for
the prepared silica magnetic particles having a spherical form are
shown in FIG. 1. It can be confirmed from FIG. 2 that the size of
the silica magnetic particles having a spherical form prepared in
example 1 is 1.56 .mu.m in average. Further, it can be confirmed
that the form thereof is a spherical form and it can be observed
that the iron oxide magnetic particles are included inside the
silica. The zeta potential of the prepared silica magnetic
particles having a spherical form is measured as -39.8 mV as shown
in FIG. 3, such that it can be confirmed that a hydroxyl group
exists on the silica surface.
Example 2
Preparing of Silica Magnetic Particles Having a Spherical Form
Using Palmitic Acid
[0082] The silica magnetic particles having a spherical form was
prepared by being performed under the same conditions as Example 1
except for using palmitic acid solution prepared by dissolving 6 g
of palmitic acid in 40 ml of cyclohexane, instead of the myristic
acid solution.
[0083] The zeta potential of the prepared silica magnetic particles
is measured as -33.3 mV, such that it can be confirmed that the
hydroxyl group exists on the silica surface.
Example 3
Preparing of Silica Magnetic Particles Having a Spherical Form
Using Stearic Acid
[0084] The silica magnetic particles having a spherical form was
prepared by being performed under the same conditions as Example 1
except for using stearic acid solution prepared by dissolving 6.8 g
of stearic acid in 40 ml of cyclohexane, instead of the myristic
acid solution.
[0085] The zeta potential of the prepared silica magnetic particles
is measured as -47.0 mV, such that it can be confirmed that the
hydroxyl group exists on the silica surface.
Example 4
Preparing of Silica Magnetic Particles Having a Spherical Form
Using Lauric Acid
[0086] The silica magnetic particles having a spherical form was
prepared by being performed under the same conditions as Example 1
except for using lauric acid solution prepared by dissolving 4.8 g
of lauric acid in 40 ml of cyclohexane, instead of the myristic
acid solution.
[0087] Analysis results of a scanning electron microscope (SEM) for
the prepared silica magnetic particles are shown in FIG. 4. It can
be confirmed that the size of the prepared silica magnetic
particles having a spherical form is about 1 to 10 .mu.m and the
form thereof is a spherical form. Further, it can be observed that
the iron oxide magnetic particles (average particle size: 300 nm)
are included inside the silica having a spherical form. The zeta
potential of the prepared silica magnetic particles is measured as
-48.1 mV, such that it can be confirmed that the hydroxyl group
exists on the silica surface.
Example 5
Preparing of Silica Magnetic Particles Having a Spherical Form
Using Oleic Acid
[0088] The silica magnetic particles having a spherical form was
prepared by being performed under the same conditions as Example 1
except for using oleic acid solution prepared by dissolving 6.8 g
oleic acid in 40 ml cyclohexane, instead of the myristic acid
solution.
[0089] Analysis results of a scanning electron microscope (SEM) for
the prepared silica magnetic particles are shown in FIG. 5. It can
be confirmed that the size of the prepared silica magnetic
particles having a spherical form is about 1 to 10 .mu.m and the
form thereof is a spherical form and uniform. The zeta potential of
the prepared silica magnetic particles is measured as -30.1 mV,
such that it can be confirmed that the hydroxyl group exists on the
silica surface.
Example 6
Preparing of Silica Magnetic Particles Having a Spherical Form
Using Myristic Acid (Scale Up)
[0090] 1200 ml of cyclohexane is put in a 2 L flask and 144 ml of
polyoxyethylene nonylphenol ether (Dongnam Chem., Korea, MONOPOL NP
1018, number average molecule weight (Mn): 585) that is surfactant
and 48 mL of sodium silicate aqueous solution (1.5M concentration)
are put therein.
[0091] Thereafter, it is dispersed for 30 minutes using an
ultrasonic wave. 0.6 g of iron oxide magnetic particles (magnetite)
(SEAHAN, Korea, SMT-02H, average particle size: 300 nm) is put in
the solution and dispersed for 30 minutes using an ultrasonic wave.
The silica magnetic particles having a spherical form was prepared
by being performed under the same conditions as Example 1 except
for dissolving 16.5 g of myristic acid in 80 ml of cyclohexane and
slowly adding this solution for 30 minutes while agitating it in a
reactor having the emulsion solution.
[0092] Analysis results of a scanning electron microscope (SEM) for
the prepared silica magnetic particles having a spherical form are
shown in FIG. 6. It can be confirmed that the size of the prepared
silica magnetic particles is 1 to 20 .mu.m. Further, it can be
confirmed that the form thereof is a spherical form and it can be
observed that the iron oxide magnetic particles (average particle
size: 300 nm) are included inside the silica. The zeta potential of
the prepared silica magnetic particles is measured as -33.9 mV,
such that it can be confirmed that a hydroxyl group exists on the
silica surface.
Comparative Example 1
Preparing of Silica Magnetic Particles Having a Spherical Form
Using Ammonium Sulfate Aqueous Solution
[0093] 800 ml of cyclohexane is put in a 1 L flask and 64 ml of
polyoxyethylene nonylphenol ether that is surfactant and 16 mL of
sodium silicate aqueous solution (1.5M concentration) are put
therein. Thereafter, it is dispersed for 30 minutes using an
ultrasonic wave. 0.6 g of iron oxide magnetic particles (magnetite)
(SEAHAN, Korea, SMT-02H, average particle size: 300 nm) is put in
the solution and dispersed for 30 minutes using an ultrasonic
wave.
[0094] An agitator is installed and the solution is agitated at
room temperature. 16 ml of Ammonium sulfate aqueous solution (1.5M
concentration) is added in the reactor while agitating the
solution. The reaction is sufficiently performed by agitating it
for 2 hours or more at room temperature to obtain the silica
magnetic particles. After the reaction completes, products in the
reactor are separated by a filter and washed twice using ethanol
and ultra pure water. The obtained silica magnetic particles are
put in a drier and dried at 120.degree. C. for 2 hours.
[0095] Analysis results of a scanning electron microscope (SEM) for
the prepared silica magnetic particles having a spherical form are
shown in FIG. 7. It can be confirmed that the size of the prepared
silica magnetic particles is about 1 to 10 .mu.m. However, it can
be appreciated that the size distribution of the silica magnetic
particles is non-uniform as compared to Example 1.
Example 7
Preparing of Silica Magnetic Particles Having a Spherical Form into
Which Amine Group is Introduced
[0096] A 250 mL flask is prepared and the inside of the flask is
substituted with nitrogen. 100 ml of octadecene (Aldrich) is put in
the flask and the silica magnetic particles having a spherical form
of 1 g prepared in Example 1 are added to the reaction flask.
Thereafter, it is dispersed for 1 hour using an ultrasonic wave.
The silica magnetic particles having a spherical form prepared in
Example 1 are dispersed, the flask is mounted on a mantle, and
aminopropyltriethoxysilane (Aldrich, USA) of 500 ul is added
thereto.
[0097] Thereafter, it reacts at 80.degree. C. for 1 hour. If the
reaction completes, it is washed three times using methanol and
ultra pure water.
[0098] The zeta potential of the prepared silica magnetic particles
having a spherical form is measured as +20.0 mV as shown in FIG. 8,
such that it can be confirmed that an amine group exists on the
silica surface.
Example 8
Preparing of Silica Magnetic Particles Having a Spherical Form into
Which Carboxylic Acid Group is Introduced
[0099] A 250 mL flask is prepared and the inside of the flask is
first substituted with nitrogen. 100 ml of octadecene (Aldrich) is
put in the flask and the silica magnetic particles having a
spherical form of 1 g introduced with the amine group prepared in
Example 7, are added to the reaction flask and dispersed for 1 hour
using an ultrasonic wave. And, 4.9 g of succinic anhydride
(Aldrich) is added to 20 ml of octadcene and dissolved for 3 hours
using an ultrasonic wave. Succinic anhydrie solution is added to
the flask in which the silica magnetic particles having a spherical
form introduced with the amine group are dispersed and the reaction
flask is then mounted on a mantle. Thereafter, it reacts at
80.degree. C. for 1 hour. If the reaction completes, it is washed
three times using methanol and ultra pure water.
[0100] The zeta potential of the prepared silica magnetic particles
having a spherical form into which the prepared carboxylic acid is
introduced is measured as -49.9 mV, such that it can be confirmed
that the carboxylic acid group exists on the silica surface.
Example 9
Preparing of Silica Magnetic Particles Having a Spherical Form into
Which Epoxy Group is Introduced
[0101] A 250 mL flask is prepared and the inside of the flask is
first substituted with nitrogen. 100 ml of octadecene (Aldrich) is
put in the flask and the silica magnetic particles having a
spherical form of 1 g prepared in Example 1 are added to the
reaction flask and dispersed for 1 hour using an ultrasonic wave.
After the silica magnetic particles having a spherical form, the
flask is mounted on a mantle and 200 ul of 3-glycidoxypropyl
trimethoxysilane (Aldrich, USA) is added and reacts at 80 for 1
hour. If the reaction completes, it is washed three times using
methanol and ultra pure water.
[0102] In the silica magnetic particles having a spherical form
into which the prepared epoxy group is introduced, since the epoxy
group does not have potential, zeta potential does not appear. In
order to confirm this, when measuring the zeta potential by
reacting 4 ml polyethyleneimine (PEI; MW=600, Alfa) that reacts
with the epoxy group, the zeta potential is measured as +17.3 mV,
such that it can be confirmed that the epoxy group exists on the
silica surface.
Example 10
Preparing of Silica Magnetic Particles Having a Spherical Form into
Which (C18) Alkyl Group is Introduced
[0103] A 250 mL flask is prepared and the inside of the flask is
first substituted with nitrogen. 100 ml of octadecene (Aldrich) is
put in the flask and the silica magnetic particles having a
spherical form of 1 g prepared in Example 1, are added to the
reaction flask and dispersed for 1 hour using an ultrasonic wave.
After the silica magnetic particles having a spherical form, the
flask is mounted on a mantle and 500 ul of
trimethoxyoctadecylsilane (Aldrich) is added and reacts at 80 for 1
hour. If the reaction completes, it is washed three times using
methanol and ultra pure water. The silica magnetic particles having
a spherical form into which the prepared (C18) alkyl group is
introduced does not exhibit zeta potential since the (C18) alkyl
group does not have potential.
Example 11
Preparing of Silica Magnetic Particles Having a Spherical Form into
Which Streptavidin Group is Introduced
[0104] A 250 mL flask is prepared and the inside of the flask is
first substituted with nitrogen. 100 ml sodium carbonate aqueous
solution (100 mM concentration) of pH 11 is put in the flask and
the silica magnetic particles having a spherical form of 1 g
introduced with the epoxy group prepared in Example 9 is added to
the reaction flask. Thereafter, it is dispersed for 1 hour using an
ultrasonic wave. After 0.5 mg streptavidin is added to the flask
where the silica magnetic particles having a spherical form
introduced with the epoxy group is dispersed, the reaction flask is
mounted on a mantle and reacts at 60 for 1 hour. If the reaction
completes, it is washed three times using methanol and ultra pure
water. In order to achieve stability, the silica magnetic particles
into which the prepared streptavidin group is introduced are
dispersed in a phosphate buffered saline.
[0105] The zeta potential of the silica magnetic particles having a
spherical form into which the prepared streptavidin group is
introduced is measured as -42.9 mV, such that it can be confirmed
that the streptavidin group exists on the silica surface.
Example 12
Preparing of Silica Magnetic Particles Having a Spherical Form into
Which Biotin Group is Introduced
[0106] A 250 mL flask is prepared and the inside of the flask is
first substituted with nitrogen. 50 ml of dichloromethane (Aldrich)
is put in the flask and the silica magnetic particles having a
spherical form of 1 g introduced with the amine group prepared in
Example 7 is added to the reaction flask and dispersed for 1 hour
using an ultrasonic wave. And, 1.0 g of biotin (ESUNG CHEMICALS
CO., LTD, Korea) is added to 100 ml of dichloromethane to prepare
biotin solution. After the biotin solution and 0.5 ml of
diisoprpylcarbodiimide (Acros) are added to the flask where the
silica magnetic particles having a spherical form is dispersed, the
reaction flask is mounted on a mantle and reacts at 60.degree. C.
for 2 hours. If the reaction completes, it is washed three times
using methanol and ultra pure water. The silica magnetic particles
having a spherical form into which the prepared biotin group is
introduced does not exhibit zeta potential since the biotin does
not have potential.
Example 13
Preparing of Silica Magnetic Particles Having a Spherical Form into
Which Iminodiacetic Acid Group is Introduced
[0107] A 250 mL flask is prepared and the inside of the flask is
first substituted with nitrogen. 100 ml sodium carbonate aqueous
solution (100 mM concentration) of pH 11 is put in the flask and
the silica magnetic particles having a spherical form of 1 g
introduced with the epoxy group prepared in Example 9 is added to
the reaction flask. Thereafter, it is dispersed for 1 hour using an
ultrasonic wave. 0.5 g of iminodiacetonitrile (Aldrich) is added to
10 ml sodium carbonate solution to prepare iminodiacetonitrile
solution. After the iminodiacetonitrile (Aldrich) solution is added
to the flask where the silica magnetic particles introduced with
the epoxy group are dispersed, the reaction flask is mounted on a
mantle and reacts at 60 for 1 hour. If the reaction completes, it
is washed three times using methanol and ultra pure water.
[0108] The zeta potential of the silica magnetic particles having a
spherical form into which the prepared iminodiacetic acid group is
introduced is measured as -38.3 mV, such that it can be confirmed
that the iminodiacetic acid group exists on the silica surface.
Example 14
Separating Nucleic Acid Using Silica Magnetic Particles Having a
Spherical Form
[0109] In order to analyze separation efficiency of the prepared
silica magnetic particles having a spherical form and the nucleic
acid, the following experiment is performed.
[0110] After the prepared standard plasmid nucleic acid (standard
nucleic acid separated from a colon bacillus cell culture media
into which pGEM T-easy vector is introduced) of 2 ug is put in a
1.5 ml tube and the silica magnetic particles of Examples 1, 4, 5,
and 6, the silica magnetic particles prepared in Comparative
Example 1 for the purpose of comparison, Promega products (MagneSil
PMPs, Cat. No. MD1360), and QIAGEN products (EZ DNA Blood 350 ul
Kit, Cat. No. 951054) are each put therein by 10 mg, 500 ul
reaction solution (4M concentration guanidine hydrochloride, 0.8M
concentration potassium acetate (Ph 4.2)) is put therein and they
are mixed well. They are left at room temperature for 5 minutes so
that the silica magnetic particles and the plasmid nucleic acid can
be bonded well. The silica magnetic particles are separated from
supernatant using a neodymium magnet and the supernatant is
completely removed using a micro pipette. 1 ml of washing solution
(80% ethanol) is put therein and is mixed well. The silica magnetic
particles are separated from supernatant using the magnet and the
supernatant is completely removed using the micro pipette. The same
washing process is repeated once more. In order to remove the
remaining washing solution, it is dried at 60.degree. C. for 5
minutes.
[0111] 100 .mu.l of ultra pure water is added to the completely
dried silica magnetic particles and is mixed well. They are left at
room temperature for 5 minutes so that the silica magnetic
particles and the plasmid nucleic acid can be eluted well. The
silica magnetic particles are separated using the magnet and the
supernatant is acquired by the micro pipette and then transported
and put in a new 1.5 ml tube. The separation yield of the plasmid
nucleic acid is calculated using ultraviolet absorption
spectrometer.
[0112] The results are listed in the following Table 1.
TABLE-US-00001 TABLE 1 Seperation yield of nucleic acid using
various silica magnetic particles Comparative Silica magnetic
Example Example Example Example Example particles 1 4 5 6 1 Promega
QIAGEN Separation 47% 53% 48% 43% 37% 32% 34% yield
[0113] As can be appreciated from Table 1, the silica magnetic
particles according to Examples of the present invention exhibit
excellent separation yield of nucleic acid as compared to
Comparative Example and marketed products.
[0114] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *